Cystic fibrosis transmembrane conductance regulator gene mutations

ABSTRACT

The present invention provides novel mutations of the CFTR gene related to cystic fibrosis or to conditions associated with cystic fibrosis. Also provided are probes for detecting the mutant sequences. Methods of identifying if an individual has a genotype containing one or more mutations in the CFTR gene are further provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/358,322, filed Jan. 25, 2012, (now U.S. Pat. No. 8,753,817), which isa divisional of U.S. application Ser. No. 12/847,960, filed Jul. 30,2010, which is a divisional of U.S. application Ser. No. 11/938,138,filed Nov. 9, 2007 (now U.S. Pat. No. 7,820,388), which is a divisionalof U.S. application Ser. No. 11/615,645, filed Dec. 22, 2006 (now U.S.Pat. No. 7,794,937), each of which is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to novel cystic fibrosis transmembraneregulator (CFTR) gene mutations and to methods for detecting thepresence of these mutations in individuals.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Cystic fibrosis (CF) is the most common severe autosomal recessivegenetic disorder in the Caucasian population. It affects approximately 1in 2,500 live births in North America (Boat et al., The Metabolic Basisof Inherited Disease, 6^(th) ed., pp 2649-2680, McGraw Hill, NY (1989)).Approximately 1 in 25 persons of northern European Caucasian descent arecarriers of the disease. The responsible gene has been localized to a250,000 base pair genomic sequence present on the long arm of chromosome7. This sequence encodes a membrane-associated protein called the“cystic fibrosis transmembrane regulator” (or “FTR”). There are greaterthan 1000 different mutations in the CFTR gene, each having varyingfrequencies of occurrence in different populations, presently reportedto the Cystic Fibrosis Genetic Analysis Consortium. These mutationsexist in both the coding regions (e.g., ΔF508, a mutation found on about70% of CF alleles, represents a deletion of a phenylalanine at residue508) and the non-coding regions (e.g., the 5T, 7T, and 9T variantscorrespond to a sequence of 5, 7, or 9 thymidine bases located at thesplice branch/acceptor site of intron 8) of the CFTR gene.

The major symptoms of cystic fibrosis include chronic pulmonary disease,pancreatic exocrine insufficiency, and elevated sweat electrolytelevels. The symptoms are consistent with cystic fibrosis being anexocrine disorder. Although recent advances have been made in theanalysis of ion transport across the apical membrane of the epitheliumof CF patient cells, it is not clear that the abnormal regulation ofchloride channels represents the primary defect in the disease.

A variety of CFTR gene mutations are known. The identification ofadditional mutations will further assist in the diagnosis of cysticfibrosis.

SUMMARY OF THE INVENTION

The inventors have discovered new mutations in the CFTR gene. Thesemutations, include 3443A>T, 2443delA (A at position 2443 is deleted),2777insTG (TG are inserted at position 2777), 3123-3125delGTT (GTT atpositions 3123-3125 are deleted) 4177delG (G at position 4177 isdeleted) 630delG (G at position 630 is deleted), 2068G>T, 1342-2A>G (Ain the splice acceptor site of intron 8, 2 nucleotides upstream ofposition 1342, is substituted with G), 297-1G>A (G in the spliceacceptor site of intron 2, 1 nucleotide upstream of position 297, issubstituted with A) 3500-2A>T (A in the splice acceptor site of intron17b, 2 nucleotides upstream of position 3500, is substituted with T),4375-2A>G (A in the splice acceptor site of intron 23, 2 nucleotidesupstream of position 4375, is substituted with G), 3172-3174delTAC (TACat positions 3172 to 3174 are deleted), 2902G>C, 4115T>C, 4185G>C,520C>G, 842A>C, 4528G>T, 448A>G, 574A>T, 3704T>C, 1248+5T>C (T in thesplice donor site of intron 7, 5 nucleotides downstream of position1248, is substituted with C), 296+12T>G (T in intron 2, 12 nucleotidesdownstream of position 296, is substituted with G), 3849+3G>A (G in thesplice donor site of intron 19, 3 nucleotides downstream of position3849, is substituted with A), 497A>G, −141C>A, 2875G>C, 2689A>G,3039A>G, 405G>C, 886G>A, 4445G>A, −228G>C, −295C>T, −379delC (C atposition −379 is deleted), and −540A>G, are related to the function ofthe CFTR gene and, therefore, to cystic fibrosis. These mutations areassociated with cystic fibrosis or are associated with conditionsassociated with cystic fibrosis. By “conditions associated with cysticfibrosis” is meant any clinical symptoms that may be found in a cysticfibrosis patient and are due to one or more CF mutations.

Accordingly, in one aspect, the present invention provides a method ofdetermining if a CFTR gene contains one or more mutations selected fromthe group consisting of 3443A>T 2443delA (A at position 2443 is deleted)2777insTG (TG are inserted at position 2777) 3123-3125delGTT (GTT atpositions 3123-3125 are deleted), 4177delG (G at position 4177 isdeleted) 630delG (G at position 630 is deleted), 2068G>T, 1342-2A>G (Ain the splice acceptor site of intron 8, 2 nucleotides upstream ofposition 1342, is substituted with G), 297-1G>A (G in the spliceacceptor site of intron 2, 1 nucleotide upstream of position 297, issubstituted with A) 3500-2A>T (A in the splice acceptor site of intron17b 2 nucleotides upstream of position 3500, is substituted with T),4375-2A>G (A in the splice acceptor site of intron 23, 2 nucleotidesupstream of position 4375, is substituted with G), 3172-3174delTAC (TACat positions 3172 to 3174 are deleted) 2902G>C, 4115T>C, 4185G>C 520C>G,842A>C 4528G>T, 448A>G, 574A>T, 3704T>C, 1248+5T>C (T in the splicedonor site of intron 7, 5 nucleotides downstream of position 1248, issubstituted with C), 296+12T>G (T in intron 2, 12 nucleotides downstreamof position 296, is substituted with G), 3849+3G>A (G in the splicedonor site of intron 19, 3 nucleotides downstream of position 3849, issubstituted with A), 497A>G, −141C>A, 2875G>C, 2689A>G, 3039A>G, 405G>C,886G>A, 4445G>A, −228G>C, −295C>1, −379delC (C at position −379 isdeleted), and −540A>G, comprising determining whether CFTR nucleic acidcontains one or more of said mutations.

In another aspect, the present invention provides a method ofidentifying if an individual has one or more mutations in the CFTR genecomprising determining if nucleic acid from the individual has one ormore mutations in one or both CFTR genes, the mutations selected fromthe group consisting of 3443A>T, 2443delA (A at position 2443 isdeleted), 2777insTG (TG are inserted at position 2777), 3123-3125delGTT(GTT at positions 3123-3125 are deleted) 4177delG (G at position 4177 isdeleted), 630delG (G at position 630 is deleted), 2068G>T, 1342-2A>G (Ain the splice acceptor site of intron 8, 2 nucleotides upstream ofposition 1342, is substituted with G), 297-1G>A (G in the spliceacceptor site of intron 2, 1 nucleotide upstream of position 297, issubstituted with A) 3500-2A>T (A in the splice acceptor site of intron17b 2 nucleotides upstream of position 3500, is substituted with T),4375-2A>G (A in the splice acceptor site of intron 23, 2 nucleotidesupstream of position 4375, is substituted with G) 3172-3174delTAC (TACat positions 3172 to 3174 are deleted), 2902G>C, 4115T>C, 4185G>C,520C>G, 842A>C, 4528G>T, 448A>G, 574A>T, 3704T>C, 1248+5T>C (T in thesplice donor site of intron 7, 5 nucleotides downstream of position1248, is substituted with C), 296+12T>G (T in intron 2, 12 nucleotidesdownstream of position 296, is substituted with G), 3849+3G>A (G in thesplice donor site of intron 19, 3 nucleotides downstream of position3849, is substituted with A), 497A>G, −141C>A, 2875G>C, 2689A>G,3039A>G, 405G>C, 886G>A, 4445G>A, −228G>C, −295C>T, −379delC (C atposition −379 is deleted), and −540A>G.

In yet another aspect, the present invention provides a method ofdetermining if an individual is predisposed to cystic fibrosis or to acondition associated with cystic fibrosis comprising determining ifnucleic acid from the individual has one or more mutations in one orboth CFTR genes, the mutations selected from the group consisting of3443A>T, 2443delA (A at position 2443 is deleted), 2777insTG (TG areinserted at position 2777), 3123-3125delGTT (GTT at positions 3123-3125are deleted), 4177delG (G at position 4177 is deleted), 630delG (G atposition 630 is deleted), 2068G>T, 1342-2A>G (A in the splice acceptorsite of intron 8, 2 nucleotides upstream of position 1342, issubstituted with G), 297-1G>A (G in the splice acceptor site of intron2, 1 nucleotide upstream of position 297, is substituted with A)3500-2A>T (A in the splice acceptor site of intron 17b, 2 nucleotidesupstream of position 3500, is substituted with T), 4375-2A>G (A in thesplice acceptor site of intron 23, 2 nucleotides upstream of position4375, is substituted with G) 3172-3174delTAC (TAC at positions 3172 to3174 are deleted), 2902G>C, 4115T>C, 4185G>C, 520C>G, 842A>C, 4528G>T,448A>G, 574A>T, 3704T>C, 1248+5T>C (T in the splice donor site of intron7, 5 nucleotides downstream of position 1248, is substituted with C)296+12T>G (T in intron 2, 12 nucleotides downstream of position 296, issubstituted with G), 3849+3G>A (G in the splice donor site of intron 19,3 nucleotides downstream of position 3849, is substituted with A),497A>G, −141C>A, 2875G>C, 2689A>G, 3039A>G, 405G>C, 886G>A, 4445G>A,−228G>C, −295C>T, −379delC (C at position −379 is deleted), and −540A>G.

In still a further aspect, the present invention provides a method ofcounseling an individual on the likelihood of having an offspringafflicted with cystic fibrosis or a condition associated with cysticfibrosis, comprising determining if nucleic acid from the individual hasone or more mutations in one or both CFTR genes, the mutations selectedfrom the group consisting of 3443A>T, 2443delA (A at position 2443 isdeleted), 2777insTG (TG are inserted at position 2777), 3123-3125delGTT(GTT at positions 3123-3125 are deleted), 4177delG (G at position 4177is deleted), 630delG (G at position 630 is deleted), 2068G>T, 1342-2A>G(A in the splice acceptor site of intron 8, 2 nucleotides upstream ofposition 1342, is substituted with G), 297-IG>A (G in the spliceacceptor site of intron 2, 1 nucleotide upstream of position 297, issubstituted with A) 3500-2A>T (A in the splice acceptor site of intron17b, 2 nucleotides upstream of position 3500, is substituted with T),4375-2A>G (A in the splice acceptor site of intron 23, 2 nucleotidesupstream of position 4375, is substituted with G), 3172-3174delTAC (TACat positions 3172 to 3174 are deleted) 2902G>C 4115T>C 4185G>C, 520C>G,842A>C 4528G>T, 448A>G, 574A>T, 3704T>C, 1248+5T>C (T in the splicedonor site of intron 7, 5 nucleotides downstream of position 1248, issubstituted with C), 296+12T>G (T in intron 2, 12 nucleotides downstreamof position 296, is substituted with G), 3849+3G>A (G in the splicedonor site of intron 19, 3 nucleotides downstream of position 3849, issubstituted with A), 497A>G, −141C>A, 2875G>C, 2689A>G, 3039A>G, 405G>C,886G>A, 4445G>A, −228G>C, −295C>T, −379delC (C at position −379 isdeleted), and −540A>G.

In some embodiments, the mutations are selected from the groupconsisting of 3443A>T, 2443delA (A at position 2443 is deleted)2777insTG (TG are inserted at position 2777), 3123-3125delGTT (GTT atpositions 3123-3125 are deleted), 4177delG (G at position 4177 isdeleted), 630delG (G at position 630 is deleted), 2068G>T, 1342-2A>G (Ain the splice acceptor site of intron 8, 2 nucleotides upstream ofposition 1342, is substituted with G), 297-IG>A (G in the spliceacceptor site of intron 2, 1 nucleotide upstream of position 297, issubstituted with A) 3500-2A>T (A in the splice acceptor site of intron17b, 2 nucleotides upstream of position 3500 is substituted with T),4375-2A>G (A in the splice acceptor site of intron 23, 2 nucleotidesupstream of position 4375, is substituted with G), and 3172-3174delTAC(TAC at positions 3172 to 3174 are deleted). In other embodiments themutations are selected from the group consisting of 2902G>C, 4115T>C,4185G>C, 520C>G, 842A>C, 4528G>T, 448A>G, 574A>T, 3704T>C, 1248+5T>C (Tin the splice donor site of intron 7, 5 nucleotides downstream ofposition 1248, is substituted with C), 296+12T>G (T in intron 2, 12nucleotides downstream of position 296, is substituted with G),3849+3G>A (G in the splice donor site of intron 19, 3 nucleotidesdownstream of position 3849, is substituted with A), 497A>G, and−141C>A.

In some embodiments, one or more mutations are evaluated for bothalleles of the CFTR gene in the individual. By this approach thegenotype of the individual can be determined at the position of eachmutation.

The presence of the mutation in the CFTR gene may be determined by anyof a variety of well known methods used to detect single base changes(transitions, transversions, and/or small deletions/insertions). Thus,genomic DNA may be isolated from the individual and tested for the CFmutations. In another approach, mRNA can be isolated and tested for theCF mutations. Testing may be performed on mRNA or on a cDNA copy.

Genomic DNA or cDNA may be subject to amplification by the polymerasechain reaction or related methods using primers directed to specificportions of the CFTR gene which contain a mutation to be detected. Thesequences of primers suitable for PCR amplification of portions of theCFTR gene in which contain the CF mutations are also provided.

The presence CF mutations can be determined in a nucleic acid bysequencing appropriate portions of the CFTR gene containing themutations sought to be detected. For example, each amplicon of the CFTRgene is sequenced with both M13 forward and reverse primers. In anotherapproach, CF mutations that change susceptibility to digestion by one ormore endonuclease restriction enzymes may be used to detect themutations. In another embodiment, the presence of one or more CFmutations can be determined by allele specific amplification. In yetanother embodiment, the presence of one or more CF mutations can bedetermined by primer extension. In yet a further embodiment, thepresence of one or more CF mutations can be determined byoligonucleotide ligation. In another embodiment, the presence of one ormore CF mutations can be determined by hybridization with a detectablylabeled probe containing the mutant CF sequence.

According to the invention, the presence of CF mutations can also bedetermined by analyzing the CF protein encoded by the mutated CF gene.The mutations include, for example, E1104V, deletion of L997, G646X,deletion of Y1014, D924H, I1328T, K1351N, L130V, Q237P, A1466S, I106V,I148F, M1191T, Y122C, V915L, I853V, A252T, R1438Q or frameshiftmutations.

Detection of CF mutations at the protein level can be detected by anymethod well known in the field. In one embodiment, detection of CFmutations is carried out by isolating CF protein and subjecting it toamino acid sequence determination. This may require fragmenting theprotein by proteolytic or chemical means prior to sequencing. Method ofdetermining an amino acid sequence are well known in the art.

In other embodiments, the presence of CFTR mutations is determined usingantibodies that bind specifically to a mutant CFTR protein sequence. Forexample, ELISA or other immunological assays known to a person skilledin the art can be used to detect CFTR mutations using specificantibodies for each mutation. Method of producing antibodies to specificsequence of a protein such as a mutation containing sequence are wellknown. For example, one may immunize an animal with the mutant CFTRprotein or with peptide fragments of the mutant protein containing themutant sequence. If monoclonal antibodies are produced, those specificfor the mutant sequence can be obtained by screening the antibodies fordifferential reactivity between the mutant CFTR protein and wildtypeCFTR protein. If a mutation specific polyclonal antisera is desired, onemay process the initial antisera by removing antibodies reactive withthe wildtype CFTR protein. Optionally, such antisera may be concentratedby affinity chromatography using the mutant CFTR protein. Further stepsto remove wild-type CFTR reactivity may be conducted.

Methods for developing monoclonal and polyclonal antibodies to definedepitopes of the CFTR protein have been previously described. See, e.g.,U.S. Pat. No. 5,981,714 (Cheng et al.,); Cohn et al., Biochem BiophysRes Commun. 1991 Nov. 27; 181(1):36-43; Walker et al, J Cell Sci. 1995June; 108 (Pt 6):2433-44: Klass et al. J Histochem Cytochem. 2000 June;48(6):831-7; Doucet et al., J Histochem Cytochem. 2003 September;51(9):1191-9; Carvelho-Oliveira et al. J Histochem Cytochem. 2004February; 52(2):193-203; and Mendes et al. J Cyst Fibros. 2004 August; 3Suppl 2:69-72.

The methods of the invention also may include detection of other CFmutations which are known in the art and which are described herein.

The present invention also provides oligonucleotide probes that areuseful for detecting the CF mutations. Accordingly, provided is asubstantially purified nucleic acid comprising 8-20 nucleotides fullycomplementary to a segment of the CFTR gene that is fully complementaryto a portion of the CFTR gene and encompasses a mutant CFTR sequenceselected from the group consisting of 3443A>T, 2443delA (A at position2443 is deleted) 2777insTG (TG are inserted at position 2777)3123-3125delGTT (GTT at positions 3123-3125 are deleted), 4177delG (G atposition 4177 is deleted) 630delG (G at position 630 is deleted),2068G>T, 1342-2A>G (A in the splice acceptor site of intron 8, 2nucleotides upstream of position 1342, is substituted with G) 297-1G>A(G in the splice acceptor site of intron 2, 1 nucleotide upstream ofposition 297, is substituted with A) 3500-2A>T (A in the splice acceptorsite of intron 17b, 2 nucleotides upstream of position 3500, issubstituted with T), 4375-2A>G (A in the splice acceptor site of intron23, 2 nucleotides upstream of position 4375, is substituted with G),3172-3174delTAC (TAC at positions 3172 to 3174 are deleted) 2902G>C,4115T>C 4185G>C, 520C>G 842A>C, 4528G>T, 448A>G, 574A>T, 3704T>C1248+5T>C (T in the splice donor site of intron 7, 5 nucleotidesdownstream of position 1248, is substituted with C), 296+12T>G (T inintron 2, 12 nucleotides downstream of position 296, is substituted withG), 3849+3G>A (G in the splice donor site of intron 19, 3 nucleotidesdownstream of position 3849, is substituted with A), 497A>G, −141C>A,2875G>C, 2689A>G, 3039A>G, 405G>C 886G>A, 4445G>A, −228G>C, −295C>T,−379delC (C at position −379 is deleted), and −540A>G, or acomplementary nucleic acid sequence thereof. In one embodiment, thepurified nucleic acid is no more than 50 nucleotides in length. Theinvention CF mutant probes may be labeled with a detectable label, whichmay include any of a radioisotope, a dye, a fluorescent molecule, ahapten or a biotin molecule.

In another aspect the present invention provides kits for one of themethods described herein. In various embodiments, the kits contain oneor more of the following components in an amount sufficient to perform amethod on at least one sample: one or more primers of the presentinvention, one or more devices for performing the assay, which mayinclude one or more probes that hybridize to a mutant CF nucleic acidsequence, and optionally contain buffers, enzymes, and reagents forperforming a method of detecting a genotype of cystic fibrosis in anucleic acid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing various CFTR mutations and characterizinginformation. A normal immunoreactive trypsinogen (IRT) value is 30 μg/Lor lower. An elevated IRT value is an indication of possible CFcondition. The normal range for a sweat test is a chloride value lessthan 40 mEq/L. An elevated chloride value is an indication of possibleCF condition. A normal individual has a value of greater than 480 μg/gfor a stool elastase test, while a value under 100 μg/g indicates severepancreatic insufficiency.

DETAILED DESCRIPTION OF THE INVENTION

CF mutations and exemplary PCR primer pairs for amplifying segments ofthe CFTR gene containing the mutations are shown in Table 1.

TABLE 1 CF mutations and associated amplification primers CF Mutation CFMutation Nucleotide Change CF Mutation Amino Acid Change Forward andReverse CF Mutation (HGVS Amino Acid (HGVS PCR Amplification NucleotideChange nomenclature)* Change nomenclature)* Primers 3443A > T c.3311A >T E1104V p.Glu1104Val q17be1F (SEQ ID NO: 33) and q17be1R (SEQ ID NO:34) 2443delA (A at position c.2311del frameshift p.Asn771fs q13-2e1F(SEQ ID NO: 2443 is deleted) 23) and q13-2e1R (SEQ ID NO: 24) 2777insTG(TG are c.2644_2645dup frameshift p.Trp882fs q14be2F (SEQ ID NO:inserted at position 2777) 27) and q14be2R (SEQ ID NO: 28) 3123 −3125delGTT (GTT c.2991_2993del deletion of p.Leu997del q17ae1F (SEQ IDNO: at positions 3123 − 3125 are L997 31) and q17ae1R (SEQ deleted) IDNO: 32) 4177delG (G at position c.4045del frameshift p.Gly1349fs q22e1F(SEQ ID NO: 4177 is deleted) 39) and q22e1R (SEQ ID NO: 40) 630delG (Gat position c.498del frameshift p.Lys166fs g5e3F (SEQ ID NO: 11) 630 isdeleted) and g5e4R (SEQ ID NO: 12) 2068G > T c.1936G > T G646X p.Gly646Xq13-1e1F (SEQ ID NO: 21) and q13-1e2R (SEQ ID NO: 22) 1342 − 2A > G (Ain the c.1210 − 2A > G splicing g9e9F (SEQ ID NO: 19) splice acceptorsite of and intron 8, 2 nucleotides g9e11R (SEQ ID NO: upstream ofposition 1342, 20) is substituted with G) 297 − 1G > A (G in the splicec.165 − 1G > A splicing s3e1F (SEQ ID NO: 7) acceptor site of intron 2,1 and nucleotide upstream of s3e2R (SEQ ID NO: 8) position 297, issubstituted with A) 3500 − 2A > T (A in the c.3368 − 2A > T splicingq18e1F (SEQ ID NO: splice acceptor site of 35) and intron 17b, 2nucleotides q18e1R (SEQ ID NO: upstream of position 36) 3500, issubstituted with T) 4375 − 2A > G (A in the c.4243 − 2A > G splicingq24e1F (SEQ ID NO: splice acceptor site of 41) and intron 23, 2nucleotides q24e1R (SEQ ID NO: upstream of position 4375, 42) issubstituted with G) 3172 − 3174delTAC (TAC c.3040_3042del deletion ofp.Tyr1014del q17ae1F (SEQ ID NO: at positions 3172 to 3174 Y1014 31) andq17ae1R (SEQ are deleted) ID NO: 32) 2902G > C c.2770G > C D924Hp.Asp924His q15e3F (SEQ ID NO: 29) and q15e4R (SEQ ID NO: 30) 4115T > Cc.3983T > C I1328T p.Ile1328Thr q22e1F (SEQ ID NO: 39) and q22e1R (SEQID NO: 40) 4185G > C c.4053G > C K1351N p.Lys1351Asn q22e1F (SEQ ID NO:39) and q22e1R (SEQ ID NO: 40) 520C > G c.388C > G L130V p.Leu130Valq4e1F (SEQ ID NO: 9) and q4e1R (SEQ ED NO: 10) 842A > C c.710A > C Q237Pp.Gln237Pro q6ae1F (SEQ ID NO: 13) and q6ae1R (SEQ ID NO: 14) 4528G > Tc.4396G > T A1466S p.Ala1466Ser q24e1F (SEQ ID NO: 41) and q24e1R (SEQID NO: 42) 448A > G c.316A > G 1106V p.Ile106Val q4e1F (SEQ ID NO: 9)and q4e1R (SEQ ID NO: 10) 574A > T c.442A > T I148F p.Ile148Phe q4e1F(SEQ ID NO: 9) and q4e1R (SEQ ID NO: 10) 3704T > C c.3572T > C M1191Tp.Met1191Thr q19e3F (SEQ ID NO: 37) and q19e4R (SEQ ID NO: 38) 1248 +5T > C (T in the c.1116 + 5T > C q7e3F (SEQ ID NO: 17) splice donor siteof intron and 7, 5 nucleotides q7e4R (SEQ ID NO: 18) downstream ofposition 1248, is substituted with C) 296 + 12T > G (T in intron c.164 +12T > G q2e2F (SEQ ID NO: 5) 2, 12 nucleotides and downstream ofposition q2e2R (SEQ ID NO: 6) 296, is substituted with G) 3849 + 3G > A(G in the c.3717 + 3G > A q19e3F (SEQ ID NO: splice donor site of intron37) and 19, 3 nucleotides q19e4R (SEQ ID NO: downstream of position 38)3849, is substituted with A) 497A > G c.365A > G Y122C p.Tyr122Cys q4e1F(SEQ ID NO: 9) and q4e1R (SEQ ID NO: 10) −141C > A c.−274C > Aq-promoter-2-1F (SEQ ID NO: 3) and q- promoter-2-1R (SEQ ID NO: 4)2875G > C c.2743G > C V915L p.Val915Leu q15e3F (SEQ ID NO: 29) andq15e4R (SEQ ID NO: 30) 2689A > G c.2557A > G 1853V p.Ile953Val q14ae5F(SEQ ID NO: 25) and q14ae6R (SEQ ID NO: 26) 3039A > G c.2907A > G A969A2^(nd) last nucleotide in q15e3F (SEQ ID NO: exon 15; no change 29) andto p.Ala969 q15e4R (SEQ ID NO: 30) 405G > C c.273G > C G91G Lastnucleotide in s3e1F (SEQ ID NO: 7) exon 3; no change to and p.Gly91.s3e2R. (SEQ ID NO: 8) 886G > A c.754G > A A252T p.Ala252Thr q6be2F (SEQID NO: 15) and q6be2R (SEQ ID NO: 16) 4445G > A c.4313G > A R1438Qp.Arg1438Gln q24e1F (SEQ ID NO: 41) and q24e1R (SEQ ID NO: 42) −228G > Cc.−361G > C q-promoter-2-1F (SEQ ID NO: 3) and q- promoter-2-1R (SEQ IDNO: 4) −295C > T c.−427C > T q-promoter-1-1F (SEQ ID NO: 1) and q-promoter-1-1R (SEQ ID NO: 2) −379delC (C at position −379 c.−512delCq-promoter-1-1F (SEQ is deleted) ID NO: 1) and q- promoter-1-1R (SEQ IDNO: 2) −540A > G q-promoter-1-1F (SEQ ID NO: 1) and q- promoter-1-1R(SEQ ID NO: 2) *HGVS nomenclature is based on Human Genome VariationSociety guidelines as adopted by Cystic Fibrosis Centre at the Hospitalfor Sick Children in Toronto. Canada and US Cystic Fibrosis Foundation,Bethesda, MD, USA

Further information relating to the CF mutations and the CFTR gene arefound in FIG. 1. The primers for amplifying segments of the CFTR genemay hybridize to coding or non-coding CFTR sequences under stringentconditions. Preferred primers are those that flank mutant CF sequences.

By “mutations of the CFTR gene” or “mutant CF sequence” is meant one ormore CFTR nucleic acid sequences that are associated or correlated withcystic fibrosis. The CF mutations disclosed in Table 1 may be correlatedwith a carrier state, or with a person afflicted with CF. Thus, thenucleic acid may be tested for any CF mutation described in Table 1. Thenucleic acid sequences containing CF mutations are preferably DNAsequences, and are preferably genomic DNA sequences; however, RNAsequences such as mRNA or hnRNA may also contain nucleic acid mutantsequences that are associated with cystic fibrosis.

By “carrier state” is meant a person who contains one CFTR allele thatis a mutant CF nucleic acid sequence, but a second allele that is not amutant CF nucleic acid sequence. CF is an “autosomal recessive” disease,meaning that a mutation produces little or no phenotypic effect whenpresent in a heterozygous condition with a non-disease related allele,but produces a “disease state” when a person is homozygous, i.e., bothCFTR alleles are mutant CF nucleic acid sequences.

By “primer” is meant a sequence of nucleic acid, preferably DNA, thathybridizes to a substantially complementary target sequence and isrecognized by DNA polymerase to begin DNA replication.

By “substantially complementary” is meant that two sequences hybridizeunder stringent hybridization conditions. The skilled artisan willunderstand that substantially complementary sequences need not hybridizealong their entire length. In particular, substantially complementarysequences comprise a contiguous sequence of bases that do not hybridizeto a target sequence, positioned 3′ or 5′ to a contiguous sequence ofbases that hybridize under stringent hybridization conditions to atarget sequence.

By “flanking” is meant that a primer hybridizes to a target nucleic acidadjoining a region of interest sought to be amplified on the target. Theskilled artisan will understand that preferred primers are pairs ofprimers that hybridize upstream of a region of interest, one on eachstrand of a target double stranded DNA molecule, such that nucleotidesmay be added to the 3′end of the primer by a suitable DNA polymerase.Primers that flank mutant CF sequences do not actually anneal to themutant sequence but rather anneal to sequence that adjoins the mutantsequence.

By “isolated” a nucleic acid (e.g., an RNA, DNA or a mixed polymer) isone which is substantially separated from other cellular componentswhich naturally accompany such nucleic acid. The term embraces a nucleicacid sequence which has been removed from its naturally occurringenvironment, and includes recombinant or cloned DNA isolates,oligonucleotides, and chemically synthesized analogs or analogsbiologically synthesized by heterologous systems.

By “substantially pure” a nucleic acid, represents more than 50% of thenucleic acid in a sample. The nucleic acid sample may exist in solutionor as a dry preparation.

By “complement” is meant the complementary sequence to a nucleic acidaccording to standard Watson/Crick pairing rules. A complement sequencecan also be a sequence of RNA complementary to the DNA sequence or itscomplement sequence, and can also be a cDNA.

By “coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that can be transcribed and/or translatedto produce the mRNA for and/or the polypeptide or a fragment thereof.Coding sequences include exons in a genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA. The anti-sense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

By “non-coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that is not transcribed into amino acidin vivo, or where tRNA does not interact to place or attempt to place anamino acid. Non-coding sequences include both intron sequences ingenomic DNA or immature primary RNA transcripts, and gene-associatedsequences such as promoters, enhancers, silencers, etc.

Nucleic acid suspected of containing mutant CF sequences are amplifiedusing one or more primers that flank the mutations under conditions suchthat the primers will amplify CFTR fragments containing the mutations,if present. The oligonucleotide sequences in Table 1 are useful foramplifying segments of the CFTR gene which contain the mutations in FIG.1.

The method of identifying the presence or absence of mutant CF sequenceby amplification can be used to determine whether a subject has agenotype containing one or more nucleotide sequences correlated withcystic fibrosis. The presence of a wildtype or mutant sequence at eachpredetermined location can be ascertained by the invention methods.

By “amplification” is meant one or more methods known in the art forcopying a target nucleic acid, thereby increasing the number of copiesof a selected nucleic acid sequence. Amplification may be exponential orlinear. A target nucleic acid may be either DNA or RNA. The sequencesamplified in this manner form an “amplicon.” While the exemplary methodsdescribed hereinafter relate to amplification using the polymerase chainreaction (“PCR”), numerous other methods are known in the art foramplification of nucleic acids (e.g., isothermal methods, rolling circlemethods, etc.). The skilled artisan will understand that these othermethods may be used either in place of, or together with, PCR methods.

The nucleic acid suspected of containing mutant CF sequence may beobtained from a biological sample. By “biological sample” is meant asample obtained from a biological source. A biological sample can, byway of non-limiting example, consist of or comprise blood, sera, urine,feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid,cultured cells, bone marrow sample and/or chorionic villi. Convenientbiological samples may be obtained by, for example, scraping cells fromthe surface of the buccal cavity. The term biological sample includessamples which have been processed to release or otherwise make availablea nucleic acid for detection as described herein. For example, abiological sample may include a cDNA that has been obtained by reversetranscription of RNA from cells in a biological sample.

By “subject” is meant a human or any other animal which contains a CFTRgene that can be amplified using the primers and methods describedherein. A subject can be a patient, which refers to a human presentingto a medical provider for diagnosis or treatment of a disease. A humanincludes pre and post natal forms. Particularly preferred subjects arehumans being tested for the existence of a CF carrier state or diseasestate.

By “identifying” with respect to an amplified sample is meant that thepresence or absence of a particular nucleic acid amplification productis detected. Numerous methods for detecting the results of a nucleicacid amplification method are known to those of skill in the art.

Specific primers may be used to amplify segments of the CFTR gene thatare known to contain mutant CF sequence. By amplifying specific regionsof the CFTR gene, the primers facilitate the identification of wildtypeor mutant CF sequence at a particular location of the CFTR gene. Primersfor amplifying various regions of the CFTR gene include the following:SEQ ID NO 1: (q-promoter-1-1F) TGTAAAACGACGGCCAGTcgtgtcctaagatttctgtgand SEQ ID NO 2: (q-promoter-1-1R) CAGGAAACAGCTATGACCCTTTCCCGATTCTGACTCare preferably used together as forward (F) and reverse (R) primers; SEQID NO 3: (q-promoter-2-1F) TGTAAAACGACGGCCAGTtgccaactggacctaaag and SEQID NO 4: (q-promoter-2-1R) CAGGAAACAGCTATGACCCAAACCCAACCCATACAC arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO 5: (q2e2F) TGTAAAACGACGGCCAGTcataattttccatatgccag and SEQ ID NO 6:(q2e2R) CAGGAAACAGCTATGACCTATGTfTGCTTTCTCTTCTC are preferably usedtogether as forward (F) and reverse (R) primers; SEQ ID NO 7: (s3e1F)TGTAAAACGACGGCCAGTcttgggttaatctccttgga and SEQ ID NO 8: (s3e2R)CAGGAAACAGCTATGACCATTCACCAGATTTCGTAGTC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 9: (q4e1F)TGTAAAACGACGGCCAGTaaagtcttgtgttgaaattctcagg and SEQ ID NO 10: (q4e1R)CAGGAAACAGCTATGACCCAGCTCACTACCTAATTTATGACAT are preferably used togetheras forward (F) and reverse (R) primers; SEQ ID NO 11: (g5e3F)TGTAAAACGACGGCCAGTacatttatgaacctgagaag and SEQ ID NO 12: (g5e4R)CAGGAAACAGCTATGACCCAGAATAGGGAAGCTAGAG are preferably used together asforward (F) and reverse (R) primers: SEQ ID NO 13: (q6ae1F)TGTAAAACGACGGCCAGTggggtggaagatacaatgac and SEQ ID NO 14: (q6ae1R)CAGGAAACAGCTATGACCCATAGAGCAGTCCTGGTTTTAC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 15: (q6be2F) TGTAAAACGACGGCCAGTaaaataatgcccatctgttg and SEQ ID NO 16: (q6be2R)CAGGAAACAGCTATGACCGTGGAAGTCTACCATGATAAACATA are preferably used togetheras forward (F) and reverse (R) primers: SEQ ID NO 17: (q7e3F)TGTAAAACGACGGCCAGTcttccattccaagatccc and SEQ ID NO 18: (q7e4R)CAGGAAACAGCTATGACCGCAAAGTTCATTAGAACTGATC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 19: (g9e9F)TGTAAAACGACGGCCAGTtggatcatgggccatgtgc and SEQ ID NO 20: (g9e1R)CAGGAAACAGCTATGACCAAAGAGACATGGACACCAAATTAAG are preferably used togetheras forward (F) and reverse (R) primers; SEQ ID NO 21: (q13-1e1F)TGTAAAACGACGGCCAGTcgaggataaatgatttgctcaaag and SEQ ID NO 22: (q13-1e2R)CAGGAAACAGCTATGACCTCGTATAGAGTTGATTGGATTGAGA are preferably used togetheras forward (F) and reverse (R) primers; SEQ ID NO 23: (q13-2e1F)TGTAAAACGACGGCCAGTtcctaactgagaccttacac and SEQ ID NO 24: (q13-2e1R)CAGGAAACAGCTATGACCTTCTGTGGGGTGAAATAC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 25: (q14ae5F)TGTAAAACGACGGCCAGTgtggcatgaaaccgtactgt and SEQ ID NO 26: (q14ae6R)CAGGAAACAGCTATGACCACATCCCCAAACTATCTTAA are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 27: (q14be2F)TGTAAAACGACGGCCAGTatgggaggaataggtgaaga and SEQ ID NO 28: (q14be2R)CAGGAAACAGCTATGACCTGGATTACAATACATACAAACA are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 29: (q15e3F)TGTAAAACGACGGCCAGTggttaagggtgcatgctcttc and SEQ ID NO 30: (q15e4R)CAGGAAACAGCTATGACCGGCCCTATTGATGGTGGATC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 31: (q17ae1F)TGTAAAACGACGGCCAGTacactttgtccactttgc and SEQ ID NO 32: (q17ae1R)CAGGAAACAGCTATGACCAGATGAGTATCGCACATTC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 33: (q17be1F)TGTAAAACGACGGCCAGTatctattcaaagaatggcac and SEQ ID NO 34: (q17be R)CAGGAAACAGCTATGACCGATAACCTATAGAATGCAGC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 35: (q18e1F)TGTAAAACGACGGCCAGTtagatgctgtgatgaactg and SEQ ID NO 36: (q18e1R)CAGGAAACAGCTATGACCGAAGGAAAGAAGAGATAAGG are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 37: (q19e3F)TGTAAAACGACGGCCAGTcccgacaaataaccaagtgac and SEQ ID NO 38: (q19e4R)CAGGAAACAGCTATGACCGCTAACACATTGCTTCAGGCTAC are preferably used togetheras forward (F) and reverse (R) primers: SEQ ID NO 39: (q22e1F)TGTAAAACGACGGCCAGTctgtcaaggttgtaaatagac and SEQ ID NO 40: (q22e1R)CAGGAAACAGCTATGACCAAGCAGGCATAATGATFC are preferably used together asforward (F) and reverse (R) primers; SEQ ID NO 41: (q24e1F)TGTAAAACGACGGCCAGTtattttcctttgagcctg and SEQ ID NO 42: (q24e1R)CAGGAAACAGCTATGACCGCAGAGGTAACTGTTCCAC are preferably used together asforward (F) and reverse (R) primers. These pairs of primers, which maybe used in multiplex amplifications, can amplify the regions of the CFTRgene shown in Table 2.

TABLE 2 CFTR Primer Pairs and Amplicon Characteristics Forward PrimerReverse Primer Exon/Intron Size (in base pairs) q-promoter-1-IFq-promoter-1-1R qp1 553 (SEQ ID NO: 1) (SEQ ID NO: 2) q-promoter-2-1Fq-promoter-2-1R qp2 634 (SEQ ID NO: 3) (SEQ ID NO: 4) q2e2F q2e2F exon 2323 (SEQ ID NO: 5) (SEQ ID NO: 6) s3eIF s3e2R exon 3 345 (SEQ ID NO: 7)(SEQ ID NO: 8) q4e1F q4e1R exon 4 413 (SEQ ID NO: 9) (SEQ ID NO: 10)g5e3F g5e4R intron 5 425 (SEQ ID NO: 11) (SEQ ID NO: 12) q6ae1F q6ae1Rexon 6a 334 (SEQ ID NO: 13) (SEQ ID NO: 14) q6be2F q6be2R exon 6b 341(SEQ ID NO: 15) (SEQ ID NO: 16) q7e3F q7e4R exon 7 431 (SEQ ID NO: 17)(SEQ ID NO: 18) g9e9F g9e11R exon 9 396 (SEQ ID NO: 19) (SEQ ID NO: 20)q13-1e1F q13-1e2R exon 13-1 355 (SEQ ID NO: 21) (SEQ ID NO: 22) q13-2e1Fq13-2e1R exon 13-2 584 (SEQ ID NO: 23) (SEQ ID NO: 24) q14ae5F q14ae6Rexon 14a 28 (SEQ ID NO: 25) (SEQ ID NO: 26) q14be2F q14be2R exon 14b 223(SEQ ID NO: 27) (SEQ ID NO: 28) q15e3F q15e4R exon 15 471 (SEQ ID NO:29) (SEQ ID NO: 30) q17ae1F q17ae1R exon 17a 280 (SEQ ID NO: 31) (SEQ IDNO: 32) q17be1F q17be1R exon 17b 504 (SEQ ID NO: 33) (SEQ ID NO: 34)q18e1F q18e1R exon 18 471 (SEQ ID NO: 35) (SEQ ID NO: 36) q19e3F q19e4Rexon 19 489 (SEQ ID NO: 37) (SEQ ID NO: 38) q22e1F q22e1R exon 22 446(SEQ ID NO: 39) (SEQ ID NO: 40) q24e1F q24e1R exon 24 426 (SEQ ID NO:41) (SEQ ID NO: 42)

If heterozygous polymorphism or mutation is present in one of theamplicons for exon 6b, the frameshift caused by the polymorphism ormutation will result in unreadable nucleotide sequences. Therefore, if abase change is detected in any one of these amplicons, sequencing shouldbe performed to verify the sequence of another strand using anappropriate primer. This verification sequencing can be performed usingthe same PCR cleanup product as template. The verification sequencingprimer for exon 6b is reflex6be1F (SEQ ID NO: 43):TTGATTGATTGATTGATTGATTT.

The nucleic acid to be amplified may be from a biological sample such asan organism, cell culture, tissue sample, and the like. The biologicalsample can be from a subject which includes any eukaryotic organism oranimal, preferably fungi, invertebrates, insects, arachnids, fish,amphibians, reptiles, birds, marsupials and mammals. A preferred subjectis a human, which may be a patient presenting to a medical provider fordiagnosis or treatment of a disease. The biological sample may beobtained from a stage of life such as a fetus, young adult, adult, andthe like. Particularly preferred subjects are humans being tested forthe existence of a CF carrier state or disease state.

The sample to be analyzed may consist of or comprise blood, sera, urine,feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid,cultured cells, bone marrow sample and/or chorionic villi, and the like.A biological sample may be processed to release or otherwise makeavailable a nucleic acid for detection as described herein. Suchprocessing may include steps of nucleic acid manipulation, e.g.,preparing a cDNA by reverse transcription of RNA from the biologicalsample. Thus, the nucleic acid to be amplified by the methods of theinvention may be DNA or RNA.

Nucleic acid may be amplified by one or more methods known in the artfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. The sequences amplified in this manner form an“amplicon.” In a preferred embodiment, the amplification is performed bythe polymerase chain reaction (“PCR”) (e.g., Mullis, K. et al., ColdSpring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al.,European Patent Application. 50,424; European Patent Application.84,796, European Patent Application 258,017, European PatentApplication. 237,362; Mullis, K., European Patent Application. 201,184;Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, Hi., U.S. Pat. No.4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194). Other knownnucleic acid amplification procedures that can be used include, forexample, transcription-based amplification systems or isothermalamplification methods (Malek, L. T. et al., U.S. Pat. No. 5,130,238;Davey. C. et al., European Patent Application 329,822; Schuster et al.,U.S. Pat. No. 5,169,766; Miller, H. I. et al., PCT application. WO89/06700; Kwoh, D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173(1989); Gingeras, T. R. et al., PCT application WO 88/10315; Walker, G.T, et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)).Amplification may be performed with relatively similar levels of eachprimer of a primer pair to generate a double stranded amplicon. However,asymmetric PCR may be used to amplify predominantly or exclusively asingle stranded product as is well known in the art (e.g., Poddar et al.Molec. And Cell. Probes 14:25-32 (2000)). This can be achieved for eachpair of primers by reducing the concentration of one primersignificantly relative to the other primer of the pair (e.g. 100 folddifference). Amplification by asymmetric PCR is generally linear. One ofordinary skill in the art would know that there are many other usefulmethods that can be employed to amplify nucleic acid with the inventionprimers (e.g., isothermal methods, rolling circle methods, etc.), andthat such methods may be used either in place of, or together with, PCRmethods. Persons of ordinary skill in the art also will readilyacknowledge that enzymes and reagents necessary for amplifying nucleicacid sequences through the polymerase chain reaction, and techniques andprocedures for performing PCR, are well known. The examples belowillustrate a standard protocol for performing PCR and the amplificationof nucleic acid sequences that correlate with or are indicative ofcystic fibrosis.

In another aspect, the present invention provides methods of detecting acystic fibrosis genotype in a biological sample. The methods compriseamplifying nucleic acids in a biological sample of the subject andidentifying the presence or absence of one or more mutant cysticfibrosis nucleic acid sequences in the amplified nucleic acid.Accordingly, the present invention provides a method of determining thepresence or absence of one or more mutant cystic fibrosis nucleic acidsequences in a nucleic acid containing sample, comprising: contactingthe sample with reagents suitable for nucleic acid amplificationincluding one or more pairs of nucleic acid primers flanking one or morepredetermined nucleic acid sequences that are correlated with cysticfibrosis, amplifying the predetermined nucleic acid sequence(s), ifpresent, to provide an amplified sample; and identifying the presence orabsence of mutant or wild type sequences in the amplified sample.

One may analyze the amplified product for the presence of absence of anyof a number of mutant CF sequences that may be present in the samplenucleic acid. As already discussed, numerous mutations in the CFTR genehave been associated with CF carrier and disease states. For example, athree base pair deletion leading to the omission of a phenylalanineresidue in the gene product has been determined to correspond to themutations of the CF gene in approximately 50% of Caucasian patientsaffected by CF. The table below identifies preferred CF sequences andidentifies which of the primer pairs of the invention may be used toamplify the sequence.

The CF mutations described herein also may be detected in conjunctionwith other CF mutations known in the art. Such additional CF mutationsinclude, for example, those known under symbols: 2789+5G>A: 711+1G>T:W1282X; 3120+1G>A: d1507; dF508: (F508C, 1507V, 1506V); N1303K; G542X,G551D, R553X, R560T, 1717-1G>A: R334W, R347P, 1078delT; RI 17H, I148T,621+IG>T; G85E; R1162X, 3659delC; 2184delA; A455E, (5T, 7T, 9T);3849+10kbC>T: and 1898+1G>A. Additional CF mutations were disclosed inU.S. application Ser. No. 11/074,903 filed Mar. 7, 2005, such as605G->C, I198-1203del/1204G->A (deletes TGGGCT and replaces G with A atposition 1204), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T,2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A, whichare hereby incorporated by reference in their entirety. Any and all ofthese mutations can be detected using nucleic acid amplified with theinvention primers as described herein or other suitable primers.

CF mutations in the amplified nucleic acid may be identified in any of avariety of ways well known to those of ordinary skill in the art. Forexample, if an amplification product is of a characteristic size, theproduct may be detected by examination of an electrophoretic gel for aband at a precise location. In another embodiment, probe molecules thathybridize to the mutant or wild type CF sequences can be used fordetecting such sequences in the amplified product by solution phase or,more preferably, solid phase hybridization. Solid phase hybridizationcan be achieved, for example, by attaching the CF probes to a microchip.Probes for detecting CF mutant sequences are well known in the art.

CF probes for detecting mutations as described herein may be attached toa solid phase in the form of an array as is well known in the art (see,U.S. Pat. Nos. 6,403,320 and 6,406,844). For example, the fullcomplement of 24 probes for CF mutations with additional control probes(30 in total) can be conjugated to a silicon chip essentially asdescribed by Jenison et al., Biosens Bioclectron. 16(9-12):757-63 (2001)(see also U.S. Pat. Nos. 6,355,429 and 5,955,377). Amplicons thathybridized to particular probes on the chip can be identified bytransformation into molecular thin films. This can be achieved bycontacting the chip with an anti-biotin antibody or streptavidinconjugated to an enzyme such as horseradish peroxidase. Followingbinding of the antibody (or streptavidin)-enzyme conjugate to the chip,and washing away excess unbound conjugate, a substrate can be added suchas tetramethylbenzidine (TMB) {3,3′,5,5′Tetramethylbenzidine} to achievelocalized deposition (at the site of bound antibody) of a chemicalprecipitate as a thin film on the surface of the chip. Otherenzyme/substrate systems that can be used are well known in the art andinclude, for example, the enzyme alkaline phosphatase and5-bromo-4-chloro-3-indolyl phosphate as the substrate. The presence ofdeposited substrate on the chip at the locations in the array whereprobes are attached can be read by an optical scanner. U.S. Pat. Nos.6,355,429 and 5,955,377, which are hereby incorporated by reference intheir entirety including all charts and drawings, describe preferreddevices for performing the methods of the present invention and theirpreparation, and describes methods for using them.

The binding of amplified nucleic acid to the probes on the solid phasefollowing hybridization may be measured by methods well known in the artincluding, for example, optical detection methods described in U.S. Pat.No. 6,355,429. In preferred embodiments, an array platform (see, e.g.,U.S. Pat. No. 6,288,220) can be used to perform the methods of thepresent invention, so that multiple mutant DNA sequences can be screenedsimultaneously. The array is preferably made of silicon, but can beother substances such as glass, metals, or other suitable material, towhich one or more capture probes are attached. In preferred embodiments,at least one capture probe for each possible amplified product isattached to an array. Preferably an array contains 10, more preferably20, even more preferably 30, and most preferably at least 60 differentcapture probes covalently attached to the array, each capture probehybridizing to a different CF mutant sequence. Nucleic acid probesuseful as positive and negative controls also may be included on thesolid phase or used as controls for solution phase hybridization.

Another approach is variously referred to as PCR amplification ofspecific alleles (PASA) (Sarkar, et al., 1990 Anal. Biochem. 186:64-68),allele-specific amplification (ASA) (Okayama, et al., 1989 J. Lab. Clin.Med. 114:105-113), allele-specific PCR (ASPCR) (Wu, et a., 1989 Proc.Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractorymutation system (ARMS) (Newton, et al., 1989 Nucleic Acids Res.17:2503-2516). The method is applicable for single base substitutions aswell as micro deletions/insertions. In general, two complementaryreactions are used. One contains a primer specific for the normal alleleand the other reaction contains a primer for the mutant allele (bothhave a common 2nd primer). One PCR primer perfectly matches one allelicvariant of the target, but is mismatched to the other. The mismatch islocated at/near the 3′ end of the primer leading to preferentialamplification of the perfectly matched allele. Genotyping is based onwhether there is amplification in one or in both reactions. A band inthe normal reaction only indicates a normal allele. A band in the mutantreaction only indicates a mutant allele. Bands in both reactionsindicate a heterozygote. As used herein, this approach will be referredto as “allele specific amplification.”

In yet another approach, restriction fragment length polymorphism(RFLP), which refers to the digestion pattern when various restrictionenzymes are applied to DNA, is used. RFLP analysis can be applied to PCRamplified DNA to identify CF mutations as disclosed herein.

In still another approach, wild type or mutant CF sequence in amplifiedDNA may be detected by direct sequence analysis of the amplifiedproducts. A variety of methods can be used for direct sequence analysisas is well known in the art. See, e.g., The PCR Technique: DNASequencing (eds. James Ellingboe and Ulf Gyllensten) BiotechniquesPress, 1992; see also “SCAIP” (single condition amplification/internalprimer) sequencing, by Flanigan et al. Am J Hum Genet. 2003 April;72(4):931-9. Epub 2003 Mar. 11. Direct sequencing of CF mutations isalso described in Strom et al., 2003 Genetics in Medicine 5(1):9-14.

In yet another approach for detecting wild type or mutant CF sequencesin amplified DNA, single nucleotide primer extension or “SNuPE” is used.SNuPE can be performed as described in U.S. Pat. No. 5,888,819 to Goeletet al., U.S. Pat. No. 5,846,710 to Bajaj, Piggee, C. et al. Journal ofChromatography A 781 (1997), p. 367-375 (“Capillary Electrophoresis forthe Detection of Known Point Mutations by Single-Nucleotide PrimerExtension and Laser-Induced Fluorescence Detection”); Hoogendoorn, B. etal., Human Genetics (1999) 104:89-93, (“Genotyping Single NucleotidePolymorphism by Primer Extension and High Performance LiquidChromatography”); and U.S. Pat. No. 5,885,775 to Haff et al. (analysisof single nucleotide polymorphism analysis by mass spectrometry).

Another method for detecting CF mutations include the Luminex xMAPsystem which has been adapted for cystic fibrosis mutation detection byTM Bioscience and is sold commercially as a universal bead array(Tag-It™).

Still another approach for detecting wild type or mutant CF sequences inamplified DNA is the oligonucleotide ligation assay or “OLA” or “OL”.The OLA uses two oligonucleotides which are designed to be capable ofhybridizing to abutting sequences of a single strand of a targetmolecule. One of the oligonucleotides is biotinylated, and the other isdetectably labeled. If the precise complementary sequence is found in atarget molecule, the oligonucleotides will hybridize such that theirtermini abut, and create a ligation substrate that can be captured anddetected. See e.g., Nickerson et al. (1990) Proc. Natl. Acad. Sci.U.S.A. 87:8923-8927. Landegren. U. et al. (1988) Science 241:1077-1080and U.S. Pat. No. 4,998,617.

These above approaches for detecting wild type or mutant CF sequence inthe amplified nucleic acid is not meant to be limiting, and those ofskill in the art will understand that numerous methods are known fordetermining the presence or absence of a particular nucleic acidamplification product.

In another aspect the present invention provides kits for one of themethods described herein. The kit optionally contain buffers, enzymes,and reagents for amplifying the CFTR nucleic acid via primer-directedamplification. The kit also may include one or more devices fordetecting the presence or absence of particular mutant CF sequences inthe amplified nucleic acid. Such devices may include one or more probesthat hybridize to a mutant CF nucleic acid sequence, which may beattached to a bio-chip device, such as any of those described in U.S.Pat. No. 6,355,429. The bio-chip device optionally has at least onecapture probe attached to a surface on the bio-chip that hybridizes to amutant CF sequence. In preferred embodiments the bio-chip containsmultiple probes, and most preferably contains at least one probe for amutant CF sequence which, if present, would be amplified by a set offlanking primers. For example, if live pairs of flanking primers areused for amplification, the device would contain at least one CF mutantprobe for each amplified product, or at least five probes. The kit alsopreferably contains instructions for using the components of the kit.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLES Example 1 Sample Collection and Preparation

Whole Blood: 5 cc of whole blood is collected in a lavender-top (EDTA)tube or yellow-top (ACD) tube. Green-top (Na Heparin) tubes areacceptable but less desirable. DNA is extracted from blood. 100 ng ormore DNA is prepared in TE or sterile water.

Amniotic Fluid: 10-15 cc of Amniotic Fluid is collected in a sterileplastic container.

Cultured Cells: Two T-25 culture flasks with 80-100% confluent growthmay be used.

Chorionic Villi: 10-20 mg of Chorionic Villi are collected in a sterilecontainer. 2-3 ml of sterile saline or tissue culture medium is added.

Transport: Whole Blood, Amniotic Fluid, Cultured Cells and ChorionicVilli can be shipped at room temperature (18°-26° C.). Amniotic Fluid,Cultured Cells or Chorionic Villi preferably is used withoutrefrigeration or freezing. Whole Blood and Extracted DNA can be shippedat 2°-10° C.

Storage: Whole Blood, Amniotic Fluid and Extracted DNA are stored at2°-10° C. Amniotic Fluid is stored at 2°-10° C. only after the aliquotis removed for culturing. Cultured Cells and Chorionic Villi are storedat room temperature (18°-26° C.).

Stability: Whole Blood is generally stable for 8 days at roomtemperature (18°-26° C.) or 8 days refrigerated at 2°-10° C. AmnioticFluid, Cultured Cells, and Chorionic Villi are generally processed toobtain DNA within 24 hours of receipt. Extracted DNA is stable for atleast 1 year at 2°-10° C.

Example 2 Amplification from DNA

Polymerase chain reaction (PCR) primer pairs were designed using theCFTR gene sequences in EMBL/Genbank (Accession Nos. M55106-M55131). EachPCR primer for the 32 separate PCR reactions contains either an M13forward linker sequence or an M13 reverse linker sequence as appropriateto allow universal sequence reaction priming. Individual PCR reactionsare performed in 96-well microtiter plates under the same conditions foreach amplicon. Subsequently, the PCR products are purified with theMillipore Montage™ PCR₉₆, Cleanup kit (Millipore, Bedford. MA) on aBeckman BioMek 2000 biorobot. Further details are provided in Strom etal., 2003 Genetics in Medicine 5(1):9-14.

In general, individual amplifications were prepared in a volume of 25μl, which is added to the 96 well microtiter plates. Each amplificationvolume contained 2 μl of the nucleic acid sample (generally 10-100 ng ofDNA), 19 μl of PCR-Enzyme Mix (PCR mix stock is prepared with 2.5 μl of100×PCR buffer, 0.5 μl Hot Start Taq (Qiagen Inc., Cat No. 203205), 0.5μl MgCl₂ (from 25 mM stock), PCR primers, and 0.2 μl of 25 mM dNTP).Master mix contained primers, Qiagen PCR buffer with MgCl₂, bovine serumalbumin (BSA) (New England BioLabs, Cat no. B9001B), and dNTPs (AmershamBiosciences, Cat no. 27-2032-01).

The final concentration in the PCR for MgCl₂ was 2.0 mM, for BSA was 0.8g/l, and for each dNTP was 0.2 mM. Primer final concentrations variedfrom about 1.2 μM to about 0.4 μM.

PCR was conducted using the following temperature profile: step 1: 96°C. for 15 minutes; step 2: 94° C. for 15 seconds; step 3: decrease at0.5° C./second to 56° C.; step 4: 56° C. for 20 seconds; step 5:increase at 0.3° C./second to 72° C., step 6: 72° C. for 30 seconds:step 7: increase 0.5° C./second up to 94° C.; step 8: repeat steps 2 to7 thirty three times; step 9: 72° C. for 5 minutes; step 10: 4° C. hold(to stop the reaction).

Example 3 Detection of CF Mutations

The purified PCR products were diluted to approximately 10 ng/μL andcycle sequencing reactions were performed with an ABI Prism Big Dye™Terminator v3.0 cycle sequencing reaction kit (Applied Biosystems,Foster City, Calif.) according to the manufacturer's protocol. The DNAprimers used for the sequencing reaction were M13 forward and reverseprimers. Big Dye™ Terminator reaction products were purified by ethanolprecipitation and analyzed on an ABI Prism 3100 Genetic Analyzer.Sequences obtained were examined for the presence of mutations by usingABI SeqScape v2.0 software. Both strands of DNA were sequenced.

PCR reactions, purifications, and cycle sequencing reactions wereperformed in 96-well microtiter plates using biorobots to avoid errorsintroduced by manual setups. Loading of samples onto the capillarysequencer was also automated. One plate was generally sufficient toperform the entire sequencing reaction for a single patient.Theoretically, if all reactions were successful, the entire sequencesfor a single patient could be obtained in 24-48 hours after receipt ofblood. In practice, however, one or more reactions may need to berepeated because of polymorphisms in intron 8 and 6a or failedreactions.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other embodimentsare within the following claims. In addition, where features or aspectsof the invention are described in terms of Markush groups, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup.

What is claimed is:
 1. A method of detecting a CFTR mutation,comprising: (a) contacting a biological sample comprising a CFTR nucleicacid obtained from a human with a detectably labeled nucleic acid probethat specifically hybridizes to a mutant CFTR nucleic acid comprising a4177delG deletion mutation, if present, but not to a wild-type CFTRnucleic acid, wherein the detectably labeled nucleic acid probecomprises a sequence of 8-20 nucleotides that is fully complementary toa portion of the mutant CFTR nucleic acid and comprises the 4177delGmutation: and (b) detecting the CFTR 4177delG deletion mutation when ahybrid is formed between the detectably labeled nucleic acid probe andthe mutant CFTR nucleic acid.
 2. The method of claim 1, wherein the4177delG mutation is assessed using genomic DNA or cDNA.
 3. The methodof claim 1, wherein the method comprises allele specific amplification.4. The method of claim 1, wherein the 4177deIG mutation is additionallydetected by primer extension.
 5. The method of claim 1, wherein the4177delG mutation is additionally detected by oligonucleotide ligation.6. The method of claim 1, wherein the 4177delG mutation is additionallydetected in a biological sample from the human comprising a CFTR proteinusing an antibody having a binding specificity for a mutated CFTRprotein and not to a wild-type CFTR protein.
 7. The method of claim 1,wherein the 4177deIG mutation is additionally detected by nucleic acidsequencing.